Synthesis of long-chain polyunsaturated fatty acids by recombinant cells

• US 8,071,341 B2
• Filed: 04/25/2011
• Issued: 12/06/2011
• Est. Priority Date: 04/22/2004
• Status: Active Grant

• Current Assignee: Commonwealth Scientific & Industrial Research Organisation (Commonwealth Of Australia As Represented By Department Of Industry, Innovation, Climate Change, Science, Research And Tertiary Education)
• Original Assignee: Commonwealth Scientific & Industrial Research Organisation (Commonwealth Of Australia As Represented By Department Of Industry, Innovation, Climate Change, Science, Research And Tertiary Education)
• Inventors: Singh, Surinder Pal, Robert, Stanley Suresh, Nichols, Peter David, Ellis Blackburn, Susan Irene, Zhou, Xue-Rong, Petrie, James Robertson, Green, Allan Graham
• Primary Examiner(s): FRONDA, CHRISTIAN L
• Application Number:
  US13/093,252
• Publication Number:
  US 20110201065A1
• Time in Patent Office: 225 Days
• Field of Search: None
• US Class Current: 435/134
• CPC Class Codes: A01H 5/10 Seeds
• CPC Class Codes: A23K 10/12 by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
• CPC Class Codes: A23K 10/30 from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms obtained by microbiological or biochemical processes, e.g. using yeasts or enzymes, A23K10/10
• CPC Class Codes: A23K 20/158 Fatty acids; Fats; Products containing oils or fats
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/02 Nutrients, e.g. vitamins, minerals
• CPC Class Codes: C11B 1/10 by extracting
• CPC Class Codes: C11C 3/12 by hydrogenation
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/0083 Miscellaneous (1.14.99)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12P 7/64 Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
• CPC Class Codes: C12P 7/6427 Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12P 7/6432 Eicosapentaenoic acids [EPA]
• CPC Class Codes: C12P 7/6434 Docosahexenoic acids [DHA]
• CPC Class Codes: C12P 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12Y 114/19004 DELTA8-fatty-acid desaturase (1.14.19.4)
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The present invention relates to methods of synthesizing long-chain polyunsaturated fatty acids, especially eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid, in recombinant cells such as yeast or plant cells. Also provided are recombinant cells or plants which produce long-chain polyunsaturated fatty acids. Furthermore, the present invention relates to a group of new enzymes which possess desaturase or elongase activity that can be used in methods of synthesizing long-chain polyunsaturated fatty acids.

Claims

1. A yeast cell comprising

docosapentaenoic acid and docosahexaenoic acid in an esterified form as part of triacylglycerols in the yeast cell;

a polynucleotide which encodes a Δ

5 elongase operably linked to a promoter which directs expression of the polynucleotide in the yeast cell;

a polynucleotide which encodes a Δ

4 desaturase operably linked to a promoter which directs expression of the polynucleotide in the yeast cell;

an exogenous polynucleotide which encodes a desaturase which desaturates an acyl-CoA substrate in a yeast cell; and

a total fatty acid content in the yeast cell which includes 2.5% (w/w) C20 ω

3 long-chain polyunsaturated fatty acids (LC-PUFAs) and 0.1% (w/w) docosapentaenoic acid.

2. The yeast cell of claim 1, wherein 50% of the docosapentaenoic acid in the yeast cell is incorporated into triacylglycerols.

3. The yeast cell of claim 1, wherein 50% of the docosahexaenoic acid in the yeast cell is incorporated into triacylglycerols.

4. The yeast cell of claim 1, wherein the total fatty acid content in the yeast cell comprises 0.13% (w/w) docosapentaenoic acid.

5. The yeast cell of claim 1, wherein the total fatty acid content in the yeast cell comprises at least 0.5% (w/w) docosapentaenoic acid.

6. The yeast cell of claim 1, wherein the desaturase which desaturates an acyl-CoA substrate is Δ

5 desaturase or Δ

6 desaturase.

7. The yeast cell of claim 1, wherein the desaturase which desaturates an acyl-CoA substrate is a bifunctional Δ

5 desaturase and Δ

6 desaturase.

8. The yeast cell of claim 1, further comprising a polynucleotide encoding a Δ

5 desaturase, a polynucleotide encoding a Δ

6 desaturase, and a polynucleotide encoding a Δ

6 elongase, each polynucleotide operably linked to a promoter capable of directing expression of the polynucleotide in the yeast cell.

9. The yeast cell of claim 1, further comprising a polynucleotide encoding a Δ

5 desaturase, a polynucleotide encoding a Δ

8 desaturase, and a polynucleotide encoding a Δ

9 elongase, each polynucleotide operably linked to a promoter capable of directing expression of the polynucleotide in the yeast cell.

10. The yeast cell of claim 1, comprising a C20 ω

3 long-chain polyunsaturated fatty acid which is eicosapentaenoic acid.

11. A process of preparing oil comprising

obtaining the yeast cell of claim 1;

and

extracting oil from the cell.

12. The yeast cell of claim 6, further comprising a Δ

9 elongase.

13. The yeast cell of claim 6, further comprising a Δ

6 elongase.

14. A yeast cell comprising

docosapentaenoic acid and docosahexaenoic acid in an esterified form as part of triacylglycerols in the yeast cell;

a polynucleotide which encodes a Δ

5 elongase operably linked to a promoter which directs expression of the polynucleotide in the yeast cell;

a polynucleotide which encodes a Δ

4 desaturase operably linked to a promoter which directs expression of the polynucleotide in the yeast cell;

a polynucleotide which encodes a Δ

5 desaturase operably linked to a promoter which directs expression of the polynucleotide in the yeast cell;

a polynucleotide which encodes a Δ

6 desaturase operably linked to a promoter which directs expression of the polynucleotide in the yeast cell;

a polynucleotide which encodes a Δ

6 elongase operably linked to a promoter which directs expression of the polynucleotide in the yeast cell;

an exogenous desaturase which desaturates an acyl-CoA substrate in the yeast cell; and

a total fatty acid content in the yeast cell which includes 2.5% (w/w) C20 ω

3 long-chain polyunsaturated fatty acids (LC-PUFAs) and 0.1% (w/w) docosapentaenoic acid.

15. The yeast cell of claim 14, wherein the desaturase which desaturates an acyl-CoA substrate is the Δ

6 desaturase.

16. A yeast cell comprising

docosapentaenoic acid and docosahexaenoic acid in an esterified form as part of triacylglycerols in the yeast cell;

a polynucleotide which encodes a Δ

5 elongase operably linked to a promoter which directs expression of the polynucleotide in the yeast cell;

a polynucleotide which encodes a Δ

4 desaturase operably linked to a promoter which directs expression of the polynucleotide in the yeast cell;

a polynucleotide which encodes a Δ

5 desaturase operably linked to a promoter which directs expression of the polynucleotide in the yeast cell;

a polynucleotide which encodes a Δ

8 desaturase operably linked to a promoter which directs expression of the polynucleotide in the yeast cell;

a polynucleotide which encodes a Δ

9 elongase operably linked to a promoter which directs expression of the polynucleotide in the yeast cell;

an exogenous desaturase which desaturates an acyl-CoA substrate in the yeast cell; and

a total fatty acid content in the yeast cell which includes 2.5% (w/w(C20 ω

3 long-chain polyunsaturated fatty acids (LC-PUFAs) and 0.1% (w/w) docosapentaenoic acid.

17. The yeast cell of claim 16, wherein the desaturase which desaturates an acyl-CoA substrate is the Δ

5 desaturase.

18. The yeast cell of claim 16, wherein the Δ

8 desaturase does not also have Δ

6 desaturase activity.

19. The yeast cell of claim 16, wherein the Δ

8 desaturase is able to catalyse the desaturation of both 18;

3ω

3 and 20;

3ω

3 fatty acids.

20. The yeast cell of claim 16, wherein at least one of said desaturases comprises a motif having the amino acid sequence QIEHHLF (SEQ ID NO:

45).